Streptococcus pneumoniae is the most common bacterial respiratory pathogen in human immunodeficiency virus (HIV)-infected individuals. Although vaccination with the pneumococcal polysaccharide vaccine (PPV) is recommended for all those >2 years of age infected with HIV, the response to the vaccine is less than optimal and correlates with the degree of immune suppression as measured by CD4+ T-lymphocyte count (1-10). In addition, vaccine recommendations in newly diagnosed HIV-positive persons with CD4 counts <200 and those concerning revaccination after 5 years are controversial as there is no evidence to confirm clinical or serological benefit. The poor immune response to vaccine antigens is likely related to the severe B cell dysfunction noted early in HIV disease. The viral protein, gp120, acts as a super-antigen restricted to recognition of the variable heavy chain 3 (VH3) gene segments (11). Moreover, gp120 activates proliferation and differentiation of B cells expressing the VH3 gene (11-14) resulting in progressive deletion of VH3- expressing B cells (15). Depletion of the VH3 expressing B cell population is highly significant as the VH3 gene family products encode >90% of anti-pneumococcal polysaccharide (PPS) antibodies (16-20). However, a comprehensive study, directly linking anti-PPS antibody levels, functionality and molecular structure/gene family usage has not been performed. We have modified a novel technique of single antigen-specific B cell isolation/culture allowing analysis of paired variable heavy (VH) and variable light (VL) gene usage and successfully applied this technique to PPS-specific B cells. In addition, we have recently developed a flow analysis technique that allows us to define the B cell subsets of PPS-responding B cells. This unique ability allows us to define the presence of memory B cells amongst PPS-responding B cell populations in HIV-negative, HIV-positive HAART-naive and HIV-positive HAART-treated populations. We therefore propose to perform a comprehensive study of the immune response to PPV in HIV-positive individuals. In Specific Aim 1, we propose to define the immune response to PPV in various stages of untreated HIV infection on a quantitative, functional and molecular level, using a novel, single antigen-specific B cell isolation and culture technique. In Specific Aim 2, we will test the hypothesis that individuals on long-term HAART therapy are capable of responding to the pneumococcal vaccine and investigate the nature of this response. In Specific Aim 3, we will study the percentage of PPS-specific B cells that are IgM or switched memory B cells in the HIV- negative population and compare this to HIV-positive, HAART-naive and HIV-positive HAART-treated population following primary vaccination with PPV. The proposed studies are thus unique, as they will provide a comprehensive picture of the immune response to PPV in the HAART-naive and HAART-treated HIV-positive populations. More importantly, the results of these studies could clarify controversies in the present vaccine recommendations in the HIV-infected population.